Monoclonal antibody against interleukin-13 receptor alpha 1 (il-13ralpha1)

ABSTRACT

The present invention relates generally to antibodies that bind to the Interleukin-13 receptor.alpha.1 chain (IL-13R.alpha.1) and antagonize IL-13 receptor-mediated signaling by IL-13 and/or IL-4. More particularly, the present invention provides humanized or human antibodies to mammalian and in particular IL-13R.alpha.1. These antibodies have uses in the treatment or prevention of IL-13- and/or IL-4-mediated diseases or conditions. The present invention further contemplates a method of modulating IL-13- and/or IL-4-mediated diseases or conditions by the administration of the subject antibodies. The present invention further provides an assay system useful for identifying antibodies or other agents which modulate IL-13 and/or IL-4 signaling through an IL-13 receptor complex. Accordingly, a method of screening for modulators of IL-13R.alpha.1/ligand interaction is also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/850,270, filed May 20, 2004, which is a continuation of PCTApplication No. PCT/AU03/00352, filed on Mar. 21, 2003 the entirecontent and disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to antibodies that bind to theInterleukin-13 receptor al chain (IL-13Rα1) and antagonize IL-13receptor-mediated signaling by IL-13 and/or IL-4. More particularly, thepresent invention provides humanized or human antibodies to mammalianand in particular IL-13Rα1. These antibodies have uses in the treatmentor prevention of IL-13- and/or IL-4-mediated diseases or conditions. Thepresent invention further contemplates a method of modulating IL-13-and/or IL-4-mediated diseases or conditions by the administration of thesubject antibodies. The present invention further provides an assaysystem useful for identifying antibodies or other agents which modulateIL-13 and/or IL-4 signaling through an IL-13 receptor complex.Accordingly, a method of screening for modulators of IL-13Rα1/ligandinteraction is also provided.

2. Description of the Prior Art

Bibliographic details of the publications referred to in thisspecification are also collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Interleukin-13 (IL-13) is a member of the interleukin (IL) family whosebiological effects have significant physiological implications sinceboth up- and down-regulation of activity of this cytokine in vivo couldpotentially provide pharmacological treatments for a wide range ofcommon pathologies. For this reason, amongst others, the study of IL-13and other IL molecules is of great medical importance. For example,IL-13 is strongly involved in the induction of IgE and IgG4 productionas well as the differentiation of T-helper (Th) cells into a secretory(Th2) phenotype. These immunostimulatory steps are critical in thedevelopment of atopic diseases which are a major threat to human health,such as anaphylaxis (Howard et al., Am J Hum Genet. 70(1): 230-236,2002; Noguchi et al., Hum Immunol 62(11): 1251-1257, 2001) as well asmilder conditions such as hay fever, allergic rhinitis and chronicsinusitis which, although not life-threatening, are responsible forconsiderable morbidity worldwide.

IL-13 is a mediator in the pathology of the acute and chronic stages ofasthma. During an asthma attack, its activity increases and its effectsinclude reduction of the capacity of lung epithelial cells to maintain atight barrier against inhaled particles and pathogens (Ahdieh et al., AmJ. Physiol. Cell Physiol. 281(6): C2029-2038, 2000) and promotion ofallergen-induced airway hyper-responsiveness (Morse et al., Am. J.Physiol. Lung Cell Mol. Physiol. 282(1): L44-49, 2002). In the longerterm, IL-13 promotes non-inflammatory structural changes to asthmaticairways, such as enhanced expression of mucin genes, airway damage andobstruction of the small airways (Howard et al., Am. J. Hum. Genet.70(1): 230-236, 2002; Danahay et al., Am. J. Physiol. Lung Cell Mol.Physiol. 282(2): L226-236, 2002).

Up-regulation of IL-13 activity may be beneficial in certain immunedeficiency conditions to reduce disease progression. In HIV infection,for example, a reduction in secretion by Th2 cells reducesantigen-specific immune responses (Bailer et al., J. Immunol. 162(12):7534-7542, 1999). IL-13, whose levels gradually decline in accordancewith disease progression in HIV, has been found to enhance antigenpresentation in immune deficiency conditions and to reduce de novoHIV-infection of macrophages (Bailer et al., Eur. J. Immunol. 30(5):1340-1349, 2000).

The biological effects of IL-13 are mediated by a dimeric receptorcomplex comprising the subunits IL-13Rα1 (or the NR4 subunit) andIL-4Rα. It is postulated that IL-13 binding to IL-13Rα1 triggersdimerization with IL-4Rα and activation of intracellular mediators thatinclude the Janus Kinases JAK1 and JAK2, as well as STAT6, ERK and p38(David et al., Oncogene 20(46): 6660-6668, 2001; Perez et al., J.Immunol. 168(3): 1428-1434, 2002).

IL-13 shows many overlapping biological effects with those of IL-4.IL-13 and IL-4 are related by sequence and are involved in many relatedprocesses, such as myelopoiesis and the regulation ofmonocyte/macrophage pro-inflammatory functions. For example, both IL-13and IL-4 have been shown to effect B cells in a similar fashion,up-regulating surface molecules such as MHC class II and CD23 molecules,and promoting the secretion of IgG4 and IgE.

The overlapping activities of IL-13 and IL-4 can be explained in part bytheir shared dimeric receptor complex. The Type I IL-13 receptor complexis comprised of an IL-13Rα1 and an IL-4Rα; this same receptor complex isalso the Type II IL-4 receptor complex (Callard et al., Immunology Today17(3): 108, 1996). As such, in looking to achieve therapeutic control ofthe IL-13 receptor complex by blocking cytokine mediated signaling, itmay be useful to have not only a molecule that antagonized signalingmediated by IL-13, but a molecule that antagonized signaling mediated byboth IL-13 and IL-4.

Antibodies to IL-13Rα1 may potentially act as antagonists ofIL-13-signaling through IL-13 receptor complex. International PatentPublication No. WO 97/15663 suggests antibodies to human IL-13Rα1 aspotential therapeutic agents. Gauchat et al. (Eur. J. Immunol. 28:4286-4298, 1998) reported murine antibodies to human IL-13Rα1 whichblocked interaction of a tagged IL-13 with a tagged and immobilizedsoluble IL-13Rα1. The antibodies also inhibited IL-13 binding toIL-13Rα1 in transfected HEK-293 cells. However, all of these antibodiesfailed to neutralize IL-13 induced biological activity, suggesting thatthey were not antagonists of the complete IL-13Rα1/IL-4Rα receptorcomplex. In a later paper, Gauchat et al. (Eur. J. Immunol. 30:3157-3164, 2000) reported a rat antibody, designated as C41, to murineIL-13Rα1 which bound to HEK-293 cells transfected with murine IL-13Rα1.However, C41 did not neutralize IL-13 induced biological activities.Further, C41 did not react with the soluble form of human IL-13Rα1.Akaiwa et al. (Cytokine 13: 75-84, 2001) reported an antibody thatrecognized soluble IL-13Rα1 by enzyme immunoassay and a tagged fulllength IL-13Rα1 transfected into COST cells. The antibody was used forimmunohistochemistry but there is no indication as to whether it was aneutralizing antibody.

In accordance with the present invention, antibodies are generated whichbind to the IL-13Rα1 chain, block IL-13 binding to the IL-13Rα1 chainand which antagonize IL-13 signaling through the IL-13Rα1/IL-4Rαcomplex. Such antibodies are proposed to inhibit IL-13 mediatedbiological activity. In a preferred embodiment, some antibodies of thepresent invention surprisingly antagonize signaling by both IL-13 andIL-4 through the IL-13Rα1/IL-4Rα complex.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

The present invention provides antibodies that function as IL-13Rα1antagonists and may be used for treating certain conditions induced byIL-13. The present invention also provides methods for treating theseconditions comprising administering an IL-13Rα1 antagonist to a patientafflicted with such a condition. Also provided are compositions for usein such methods comprising one or more IL-13Rα1 antagonists.

The IL-13Rα1 chain may be from any animal, including a mammal such as ahuman. Preferred IL-13Rα1 chains are the human IL-13Rα1 chain, themurine IL-13Rα1 chain, the rat IL-13Rα1 chain, the canine IL-13Rα1chain, the ovine IL-13Rα1 chain or the cynamologus monkey IL-13Rα1chain. Preferably, the IL-13Rα1 chain is the human IL-13Rα1 chain. Thereis a high level of sequence homology between IL-13Rα1 chains fromdifferent species. For example, ovine IL-13Rα1 has 87% homology at theamino acid level and 88.7% homology at the DNA level to human IL-13Rα1.Ovine IL-13Rα1 has 75% homology at the amino acid level and 82.2%homology at the DNA level to murine IL-13Rα1. Human IL-13Rα1 has 75%homology at the amino acid level and 81.3% homology at the DNA level tomurine IL-13Rα1. Consequently, the present invention contemplates anIL-13Rα1 chain or its equivalent from any source such as an IL-13Rα1having at least about 65% identity to human IL-13Rα1 after optimalalignment. The antibodies of the present invention bind, interact orotherwise associate to the IL-13Rα1 or a portion thereof. The antibodiesmay be specific for IL-13Rα1 from a particular species, such as humanIL-13Rα1, or, in view of the level of sequence similarity betweenIL-13Rα1 from different species, the antibodies may show somecross-reactivity with IL-13Rα1 from two or more species. In the case ofantibodies directed towards human IL-13Rα1, some level ofcross-reactivity with other mammalian forms of IL-13Rα1 may be desirablein certain circumstances, such as for example, for the purpose oftesting antibodies in animal models of a particular disease and forconducting toxicology studies in a manner where IL-13 and/or IL-4signaling in the test animal is affected by the test antibody. Specieswhere cross-reactivity of an antibody to human IL-13Rα1 may be desirableinclude monkey, sheep, dog and rat. Accordingly, one preferred group ofantibodies are those which exhibit some level of speciescross-reactivity. A particularly preferred group of such antibodies arethose to human IL-13Rα1 which exhibit some level of speciescross-reactivity.

Antibodies of the present invention include, but are not limited to,antibodies that bind IL-13Rα1 and inhibit IL-13 induced signalingthrough the IL-13 receptor complex, and other compounds that inhibit abiological effect that results from the binding of IL-13 to a cellsurface IL-13 receptor. A preferred group of antibodies are those thatinhibit signaling by both IL-13 and IL-4 through the IL-13 receptorcomplex.

Preferably, the antibodies are monoclonal antibodies or antigen-bindingfragments thereof. Most preferably, the antibodies are humanized orhuman antibodies suitable for administration to humans. These includehumanized antibodies prepared, for example, from murine monoclonalantibodies and human monoclonal antibodies which may be prepared, forexample, using transgenic mice or by phage display.

Antibodies in accordance with the present invention include the murinemonoclonal antibody 1D9, and humanized forms of mAb 1D9.

The present invention contemplates methods of modulating IL-13- and/orIL-4-mediated diseases or conditions by the administration of antibodiesof the present invention. Conditions to be treated in accordance withthe present invention include fibrosis, Hodgkin's disease, ulcerativecolitis, scleroderma, lung disorders such as asthma and chronicobstructive pulmonary disease, allergic rhinitis, oncologicalconditions, inflammatory bowel disease and other inflammatory conditionsin the gastrointestinal tract, allergic reactions to medication and anyother IL-13 mediated diseases or conditions.

The present invention also provides an assay system useful foridentifying antibodies or other agents which modulate IL-13 and/or IL-4signaling through an IL-13 receptor complex. Accordingly, a method ofscreening for modulators of IL-13Rα1/ligand interaction, which methodinvolves the assay system, is provided.

A hybridoma producing murine monoclonal antibody to ID9 was deposited on21 Mar. 2003 at the European Collection of Cell Cultures (ECACC), Centrefor Applied Microbiology and Research, Porton Down, Salisbury, UnitedKingdom, under Accession No. 03032101 on Mar. 21, 2003.

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 1.

TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1Nucleotide sequence encoding IL-4Rα 2 Amino acid sequence of IL-4Rα 3Nucleotide sequence encoding human IL-13Rα1 4 Amino acid sequence ofhuman IL-13Rα1 5 Nucleotide sequence encoding gp130 6 Amino acidsequence of gp130 7 Nucleotide sequence encoding IL-4Rα-gp130 fusion 8Amino acid sequence of IL-4Rα-gp 130 fusion 9 Nucleotide sequenceencoding IL-13Rα1-gp130 fusion 10 Amino acid sequence of IL-13Rα1-gp130fusion 11 IL-13Rα1 5′ oligonucleotide 12 IL-13Rα1 3′ oligonucleotide 13gp130 5′ oligonucleotide 14 gp130 3′ oligonucleotide 15 IL-4Rα 5′amplification oligonucleotide 16 IL-4Rα 3′ amplification oligonucleotide17 IL-4Rα 5′ oligonucleotide 18 IL-4Rα 3′ oligonucleotide 19 Amino acidsequence of murine 1D9 CDR1 in V_(L) domain 20 Amino acid sequence ofmurine 1D9 CDR2 in V_(L) domain 21 Amino acid sequence of murine 1D9CDR3 in V_(L) domain 22 Amino acid sequence of murine 1D9 CDR1 in V_(H)domain 23 Amino acid sequence of murine 1D9 CDR2 in V_(H) domain 24Amino acid sequence of murine 1D9 CDR3 in V_(H) domain 25 Amino acidsequence of murine 1D9 CDR regions from V_(L) domain grafted onto humanconsensus framework 26 Amino acid sequence of murine 1D9 CDR region fromV_(H) domain grafted onto human consensus framework 27 Amino acidsequence of V_(L) domain of murine 1D9 28 Amino acid sequence of V_(H)domain of murine 1D9

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation showing that dimerization ofchimeric receptors mediated by IL-13 or IL-4 induces STAT-3phosphorylation through the gp130 intracellular domain and subsequentlyexpression of the STAT-3 activated luciferase reporter gene.

FIG. 2 is a diagrammatic representation showing construction of chimericreceptors incorporating the IL-13Rα1 or IL-4Rα extracellular domain andthe transmembrane and intracellular domains of gp130; cloned into thepEFBOS vectors for expression as an N-terminal FLAG-tagged protein.

FIG. 3 is a photographic representation showing transient expression ofchimeric receptor constructs in COS cells. COS cells were transfectedwith pEFBOS encoding FLAG-tagged IL-13Rα1-130, FLAG-tagged IL-4Rα-gp130(two independent clones) or control β-gal. Cell lysates were recoveredat 72 hrs and after SDS-PAGE and Western transfer, probed with either ananti-FLAG antibody or the IL-13Rα1-specific mAb 1D9.

FIG. 4 is a graphical representation showing a dose-response analysis toLIF, IL-13 and IL-4 of chimeric receptor transfected 293A12 lines 3.1.2and 3.2.4. 293A12 cells are derivatives of 293T cells that have beenstably transfected with a STAT-3 luciferase reporter construct. Afterinitial analysis, lines 3.1.2 (A) and 3.2.4 (B) were expanded andassayed against titrating LIF, IL-13 and IL-4. Both lines and anadditional line, 3.2.5 were cloned by limiting dilution. Assayconditions were 5×10⁴ cells/well 24 hr incubation.

FIG. 5 is a graphical representation showing Biosensor analysis of mAb1D9 inhibition of binding of chimeric human IL-13Rα1-Fc to human andmouse IL-13. mAb 1D9 and the chimeric receptors were pre-incubated atthe indicated concentrations for 1 hour prior to analysis.

FIG. 6 is a graphical representation showing that mouse mAb 1D9 inhibitsthe binding of chimeric human (A) but not chimeric mouse (B) IL-13Rα1-Fcto plate bound mouse IL-13. Titrating chimeric receptor proteins werepre-incubated with mAbs (final concentration 50 μg/ml) for 45 min priorto transfer to assay plates coated with mouse IL-13. Anti-VEGF-Bspecific mAb 6C12 was used as a negative control.

FIG. 7 is a graphical representation showing analysis of furtherIL-13Rα1 specific mouse mAbs for ability to inhibit binding of chimerichuman IL-13Rα1 to plate bound mouse IL-13. Titrating chimeric humanreceptor was pre-incubated with IL-13Rα1 specific mAbs (1D9, 6A9, 3F10,2A2) or negative control antibodies (2H10, 6C12) at a finalconcentration of 50 m/ml for 45 min prior to transfer to assay plates.

FIG. 8 is a graphical representation showing that mouse mAbs against thehuman IL-13Rα1 inhibit the 3.2.4 response to IL-13. 3.2.4-cells arecultured for 24 hrs in the presence of 10 or 1 ng/ml IL-13 and theindicated concentration of mAb. mAbs 1D9, 6A9 and 2A2 are IL-13Rα1specific mAbs and 2H10 was an isotype matched negative control antibody.Percentage inhibition is calculated from (response to cytokine plusmAb/response to cytokine only)×100.

FIG. 9 is a graphical representation showing that mouse mAbs against thehuman IL-13Rα1 inhibit the 3.2.4 response to IL-4. 3.2.4-cells werecultured for 24 hrs in the presence of 10 or 1 ng/ml IL-4 and theindicated concentration of mAb. mAbs 1D9, 6A9 and 2A2 are IL-13Rα1specific mAbs and 2H10 was an isotype matched negative control antibody.Percentage inhibition is calculated from (response to cytokine plusmAb/response to cytokine only)×100.

FIG. 10 is a representation of the amino acid sequence of murine mAb ID9variable domains and human consensus framework. Sequence numbering isaccording to Kabat et al., (Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed., 1991, ed. Bethesda: Public Health Services,National Institutes of Health) and key framework residues are indicatedby bullets (Baca et al., J. Biol. Chem. 272(16): 10678-10684, 1997). CDRsequences are underlined and are defined according to the sequencedefinition of Kabat et al. (1991, supra) with the exception of CDR-H1,which is the combined sequence and structural definition (Chothia etal., Nature 342(6252): 877-883, 1989). The framework is the consensussequence for the human light chain K subgroup I-heavy chain subgroup III(Chuntharapai et al., Cytokine 15(5): 250-260, 2001). The sequencesshown correspond to the following sequence identifiers:

V_(L) Domain Mu. 1D9 SEQ ID NO: 27

V_(L) Domain HuV_(L)KI SEQ ID NO: 25

V_(H) Domain Mu. 1D9 SEQ ID NO: 28

V_(H) Domain HuV_(H)III SEQ ID NO: 26

FIGS. 11A and 11B are graphical representations of binding affinities ofthe chimeric and CDR-grafted Fab fragment. (A) Competition ELISA ofchimeric or CDR-grafted 1D9 phage displayed Fabs binding to plate boundhIL-13Rα1-Fc (ECD) (2.5 μg/ml) competed by soluble hIL-13Rα1 (ECD). (B)Biosensor competition assay of soluble 1D9 chimeric or CDR-grafted Fabbinding to immobilized hIL-13Rα1 (ECD) competed by soluble hIL-13Rα1(ECD). Fold-difference in affinity is calculated from (IC₅₀/IC₅₀).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to antibodies that bind,interact or otherwise associated to or with the IL-13Rα1 chain or afragment, portion or part thereof and antagonize IL-13 receptor-mediatedsignaling by IL-13 and/or IL-4 and which may be employed in the methodsof the present invention. The antibodies preferably are monoclonalantibodies or antigen-binding fragments thereof. Preferably, theantibodies are in isolated, homogenous or fully or partially purifiedform.

Most preferably, the antibodies are humanized or human antibodiessuitable for administration to humans. These include humanizedantibodies prepared, for example, from murine monoclonal antibodies, andhuman monoclonal antibodies which may be prepared, for example, usingtransgenic mice as described below, or by phage display.

Reference to “binding” of an antibody means binding, interacting orassociating with or to a target antigen such as IL-13Rα1. Reference to“IL-13Rα1” includes it fragments or portions which comprise the epitopesto which an antibody binds. Consequently, reference to an antibodybinding to IL-13Rα1 includes the binding, interaction or association ofthe antibody or an antigen-binding portion thereof, part, fragment orepitope-containing region thereof.

Generally, “binding”, “interaction” or “association” means or includesthe specific binding, interaction or association of the antibody to anIL-13Rα1 or a portion thereof.

The biological effects of IL-13 are mediated by a dimeric receptorcomplex comprise the subunits IL-13Rα1 (or the NR4 subunit) and IL-4Rα(referred to hereinafter as the IL-13 receptor). Thus, some antibodiesraised against IL-13Rα1 which block IL-13 binding and/or signalingthrough the IL-13 receptor complex, may also block the signaling of IL-4through the IL-13 receptor complex.

Examples of antibodies contemplated by the present invention includethose that bind to IL-13Rα1 and block the signaling of IL-13 through theIL-13 receptor complex, and preferably those that bind to IL-13Rα1 andblock the signaling of IL-13 and/or IL-4 through the IL-13 receptorcomplex, thereby inhibiting an IL-13 induced and/or an IL-4 inducedbiological activity. Such antibodies, referred to herein as blockingantibodies, may be raised with an IL-13Rα1 polypeptide or immunogenicparts thereof, such as for example, the extracellular domain of IL-13Rα1and screened in assays for the ability to block the signaling of IL-13and/or IL-4 through the IL-13 receptor complex. Suitable assays areassays that test the antibodies for the ability to inhibit binding ofIL-13 to cells expressing the IL-13 receptor complex, or that testantibodies for the ability to reduce a biological or cellular responsethat results from the signaling of IL-13 and IL-4 through the IL-13receptor complex.

In one embodiment, the present invention provides antibodies that bindto IL-13Rα1 and inhibit IL-13 signaling through the IL-13 receptorcomplex.

In a further embodiment, the present invention provides antibodies thatbind to IL-13Rα1 and inhibit IL-13- and IL-4-signaling through the IL-13receptor complex.

Preferably the antibodies are monoclonal antibodies or antigen-bindingfragments thereof.

Most preferably, the antibodies are human or humanized monoclonalantibodies suitable for use in human therapeutics.

As such, in a preferred embodiment, the present invention providesantibodies that are human or humanized monoclonal antibodies that bindto IL-13Rα1 and inhibit IL-13 signaling through the IL-13 receptorcomplex.

In an especially preferred embodiment, the present invention providesantibodies that are human or humanized monoclonal antibodies that bindto IL-13Rα1 and inhibit IL-13- and IL-4-signaling through the IL-13receptor complex.

Reference to an “antibody” or “antibodies” includes reference to all thevarious forms of antibodies, including but not limited to wholeantibodies, antibody fragments, including, for example, Fv, Fab, Fab′and F(ab′)₂ fragments, humanized antibodies, human antibodies (e.g.,produced in transgenic animals or through phage display) andimmunoglobulin-derived polypeptides produced through genetic engineeringtechniques.

Unless stated otherwise, specificity in respect of an antibody of thepresent invention is intended to mean that the antibody does not exhibitany meaningful cross-reactivity with non-IL-13Rα1 proteins. However, itis not intended to indicate that there is no cross-reactivity with otherforms of the IL-13Rα1 which may exist, (for example, soluble forms,splice variants or fragments of the receptor), nor is it intended toindicate that no cross-reactivity with IL-13Rα1 from other species mayexist. The amino acid sequence of IL-13Rα1 is a well conserved acrossspecies, with other mammalian forms of the receptor showing substantialamino acid homology with the human IL-13Rα1 chain.

The antibodies may be specific for an IL-13Rα1 chain from a particularspecies, such as human IL-13Rα1, or, because of the level sequencesimilarity between IL-13Rα1 chains from certain mammalian species, mayshow some cross-reactivity with IL-13Rα1 chains from other mammalianspecies. In the case of antibodies directed towards human IL-13Rα1, somelevel of cross reactivity with other mammalian forms of IL-13Rα1 may bedesirable in certain circumstances. For example, such antibodies areuseful for the purpose of testing antibodies in animal models of aparticular disease, and for conducting toxicology studies in a mannerwhere IL-13 and/or IL-4 signaling in the test animal is affected by thetest antibody. Species where cross reactivity of an antibody to humanIL-13Rα1 may be desirable include monkey, sheep, dog and rat.Accordingly, one preferred group of antibodies are those which exhibitsome level of species cross reactivity. A particularly preferred groupof antibodies are those antibodies to human IL-13Rα1 which exhibit somelevel of species cross-reactivity.

The antibodies of the present invention bind to the IL-13Rα1 chain. TheIL-13Rα1 chain may be the human IL-13Rα1 chain or from another animal,such as the murine IL-13Rα1 chain, the rat IL-13Rα1 chain, the canineIL-13Rα1 chain, the ovine IL-13Rα1 chain and the cynamologus monkeyIL-13Rα1 chain. Preferably, the IL-13Rα1 chain is the human IL-13Rα1chain. There is a high level of sequence homology between IL-13Rα1chains from different species. For example, the ovine IL-13Rα1 chain is87% homologous at the amino acid level and 88.7% homologous at the DNAlevel to human IL-13Rα1. Ovine IL-13Rα1 is 75% homologous at the aminoacid level and 82.2% homologous at the DNA level to murine IL-13Rα1.Human IL-13Rα1 is 75% homologous at the amino acid level and 81.3%homologous at the DNA level to murine IL-13Rα1.

In a preferred embodiment, the present invention provides antibodiesthat bind to human IL-13Rα1 and to cynamolgus monkey IL-13Rα1 andinhibit IL-13 signaling through the IL-13 receptor complex.

In a further preferred embodiment, the present invention providesantibodies that bind to human IL-13Rα1 and to ovine IL-13Rα1 and whichinhibit IL-13 signaling through the IL-13 receptor complex.

In still a further preferred embodiment, the present invention providesantibodies that bind to human IL-13Rα1 and to canine IL-13Rα1 and whichinhibit IL-13 signaling through the IL-13 receptor complex.

In yet a further preferred embodiment, the present invention providesantibodies that bind to human IL-13Rα1 and to rat IL-13Rα1 and whichinhibit IL-13 signaling through the IL-13 receptor complex.

In yet a further preferred embodiment, the present invention providesantibodies that bind to human IL-13Rα1 and to murine IL-13Rα1 and whichinhibit IL-13 signaling through the IL-13 receptor complex.

The antibodies of the present invention may be prepared by well knownprocedures. See, for example, Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Kennet et al. (eds.), Plenum Press,New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land(eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1988).

One method for producing an antibody of the present invention comprisesimmunizing a non-human animal, such as a mouse or a transgenic mouse,with an IL-13Rα1 polypeptide, or immunogenic parts thereof, such as, forexample, the extracellular domain of IL-13Rα1, whereby antibodiesdirected against the IL-13Rα1 polypeptide are generated in said animal.

Both polyclonal and monoclonal antibodies can be produced by thismethod. The methods for obtaining both types of sera are well known inthe art. Polyclonal sera are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of an IL-13Rα1 polypeptide, or immunogenic parts thereof, suchas, for example, the extracellular domain of IL-13Rα1, collecting serumfrom the animal and isolating IL-13Rα1 specific sera by any of the knownimmunoadsorbent techniques. Antibodies produced by this technique aregenerally less favoured, because of the potential for heterogeneity ofthe product.

The use of monoclonal antibodies is particularly preferred because ofthe ability to produce them in large quantities and the homogeneity ofthe product. Monoclonal antibodies may be produced by conventionalprocedures.

The present invention contemplates a method for producing a hybridomacell line comprises immunizing a non-human animal, such as a mouse or atransgenic mouse, with an IL-13Rα1 polypeptide, or immunogenic partsthereof, such as, for example, the extracellular domain of IL-13Rα1;harvesting spleen cells from the immunized animal; fusing the harvestedspleen cells to a myeloma cell line to generate hybridoma cells; andidentifying a hybridoma cell line that produces a monoclonal antibodythat binds an IL-13Rα1 polypeptide.

Such hybridoma cell lines and the anti-IL-13Rα1 monoclonal antibodiesproduced by them are encompassed by the present invention. Monoclonalantibodies secreted by the hybridoma cell lines are purified byconventional techniques. Hybridomas or the monoclonal antibodiesproduced by them may be screened further to identify monoclonalantibodies with particularly desirable properties, such as the abilityto inhibit IL-13- and IL-4-signaling through the IL-13 receptor complex.

The IL-13Rα1 polypeptide or immunogenic part thereof that may be used toimmunize animals in the initial stages of the production of theantibodies of the present invention may be from any mammalian source.Preferably, the IL-13Rα1 polypeptide or immunogenic part thereof ishuman IL-13Rα1.

Antigen-binding fragments of antibodies of the present invention may beproduced by conventional techniques. Examples of such fragments include,but are not limited to, Fab, Fab′, F(ab′) 2 and Fv fragments, includingsingle chain Fv fragments (termed sFv or scFv). Antibody fragments andderivatives produced by genetic engineering techniques, such asdisulphide stabilized Fv fragments (dsFv), single chain variable regiondomain (Abs) molecules and minibodies are also contemplated for use.Unless otherwise specified, the terms “antibody” and “monoclonalantibody” as used herein encompass both whole antibodies andantigen-binding fragments thereof.

Such derivatives of monoclonal antibodies directed against IL-13Rα1 maybe prepared and screened for desired properties, by known techniques,including the assays described herein. The assays described hereinprovide the means to identify derivatives of the antibodies of thepresent invention that bind to IL-13Rα1, as well as identify thosederivatives that also retain the activity of inhibiting signaling byIL-13 through the IL-13 receptor complex, and preferably, inhibitingsignaling by IL-13 and IL-4 through the IL-13 receptor complex. Certainof the techniques involve isolating DNA encoding a polypeptide chain (ora portion thereof) of a mAb of interest, and manipulating the DNAthrough recombinant DNA technology. The DNA may be fused to another DNAof interest, or altered (e.g. by mutagenesis or other conventionaltechniques) to add, delete, or substitute one or more amino acidresidues, for example.

DNA encoding antibody polypeptides (e.g. heavy or light chain, variableregion only or full length) may be isolated from B-cells of mice thathave been immunized with IL-13Rα1. The DNA may be isolated byconventional procedures such as polymerase chain reaction (PCR). Phagedisplay is another example of a known technique whereby derivatives ofantibodies may be prepared. In one approach, polypeptides that arecomponents of an antibody of interest are expressed in any suitablerecombinant expression system, and the expressed polypeptides areallowed to assemble to form antibody molecules.

Single chain antibodies may be formed by linking heavy and light chainvariable region (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable regionpolypeptides (VL and VH). The resulting antibody fragments can formdimers or trimers, depending on the length of a flexible linker betweenthe two variable domains (Kortt et al., Protein Engineering 10: 423,1997). Techniques developed for the production of single chainantibodies include those described in U.S. Pat. No. 4,946,778; Bird(Science 242: 423, 1988), Huston et al. (Proc. Natl. Acad. Sci. USA 85:5879, 1988) and Ward et al. (Nature 334: 544, 1989). Single chainantibodies derived from antibodies provided herein are encompassed bythe present invention.

In one embodiment, the present provides derivatives of the antibodies ofthe present invention that bind to IL-13Rα1, and inhibit signaling byIL-13 through the IL-13 receptor complex. Preferably, the derivativesblock signaling by Il-13 and IL-4 through the Il-13 receptor complex.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG1 or IgG4 monoclonal antibodies may be derived from an IgM monoclonalantibody, for example, and vice versa. Such techniques allow thepreparation of new antibodies that possess the antigen-bindingproperties of a given antibody (the parent antibody), but also exhibitbiological properties associated with an antibody isotype or subclassdifferent from that of the parent antibody. Recombinant DNA techniquesmay be employed. Cloned DNA encoding particular antibody polypeptidesmay be employed in such procedures, e.g. DNA encoding the constantregion of an antibody of the desired isotype.

The monoclonal production process described above may be used inanimals, for example mice, to produce monoclonal antibodies.Conventional antibodies derived from such animals, for example murineantibodies, are known to be generally unsuitable for administration tohumans as they may cause an immune response. Therefore, such antibodiesmay need to be subjected to a humanization process in order to provideantibodies suitable for administration to humans. Such humanizationprocesses are well known in the art and are described in further detailbelow.

Additional embodiments include chimeric antibodies and humanizedversions of murine monoclonal antibodies. Such chimeric or humanizedantibodies may be prepared by known techniques, for example, CDRgrafting, and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a chimericmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding sites (complementarity determining regionsCDRs) of a murine monoclonal antibody and a variable region fragment(lacking the antigen-binding site) derived from a human antibody.Procedures for the production of chimeric and humanized monoclonalantibodies include those described in Riechmann et al. (Nature 332: 323,1988) Liu et al. (Proc. Natl. Acad. Sci. USA 84: 3439, 1987), Larrick etal. (Bio/Technology 7: 934, 1989) and Winter and Harris (TIPS 14: 139,1993).

The complementarity determining regions (CDRs) of a given antibody maybe identified using the system described by Kabat et al. in Sequences ofProteins of Immunological Interest, 5th Ed., US Dept. of Health andHuman Services, PHS, NIH, NIH Publication No. 91-3242, 1991).

For example, the murine monoclonal antibody 1D9 has been subjected tohumanization to reduce the immunogenicity of the antibody in a targethost, as described in the Examples below. Murine monoclonal antibody 1D9has a specific and potent antagonistic effect against IL-13Rα1 andinhibits signaling through the IL-13 receptor and IL-4 signaling throughthe IL-13 receptor. However, the potential immunogenicity of mAb 1D9 inother hosts, and in particular humans, makes the use of mAb 1D9unsuitable as a therapeutic agent in these hosts.

In a particular embodiment, the antibodies of the present inventioncomprise within the variable region of their light chain, at least oneof the CDRs found in the light chain of mAb 1D9. The CDRs of mAb 1D9 aredisclosed in FIG. 10 and in SEQ ID NOs: 9-24. Thus, among the antibodiescontemplated by the present invention are those that comprise from oneto all three of the CDR sequences from the light chain variable regionof mAb 1D9. Further, among the antibodies contemplated by the presentinvention are those that comprise from one to all three of the CDRsequences from the heavy chain variable region of mAb 1D9. In apreferred embodiment, the antibodies of the present invention comprisefrom one to all six CDR sequences from the heavy and light chainvariable regions of mAb 1D9.

Procedures for generating human antibodies in non-human animals havealso been developed and are well known to those skilled in the art. Theantibodies may be partially human, or preferably completely human. Forexample, transgenic mice into which genetic material encoding one ormore human immunoglobulin chains has been introduced may be used toproduce the antibodies of the present invention. Such mice may begenetically altered in a variety of ways. The genetic manipulation mayresult in human immunoglobulin polypeptide chains replacing endogenousimmunoglobulin chains in at least some (preferably virtually all)antibodies produced by the animal upon immunization.

Mice in which one or more endogenous immunoglobulin genes have beeninactivated by various means have been prepared. Human immunoglobulingenes have been introduced into the mice to replace the inactivatedmouse genes. Antibodies produced in the animals incorporate 22 humanimmunoglobulin polypeptide chains encoded by the human genetic materialintroduced into the animal. Examples of techniques for production anduse of such transgenic animals are described in U.S. Pat. Nos.5,814,318, 5,569,825, and 5,545,806, which are incorporated by referenceherein.

As such, antibodies of the present invention may include, but are notlimited to, partially human (preferably fully human) monoclonalantibodies that inhibit signaling by IL-13, and preferably, inhibitsignaling by IL-13 and IL-4 through the IL-13 receptor complex.

Another method for generating human antibodies is phage display. Phagedisplay techniques for generating human antibodies are well known tothose skilled in the art, and include the methods used by companies suchas Cambridge Antibody Technology and MorphoSys and which are describedin International Patent Publication Nos. WO 92/01047, WO 92/20791, WO93/06213 and WO 93/11236.

Antibodies of the present invention may be employed in vitro or in vivo.Among the uses for antibodies of the present invention are assays(either in vitro or in vivo) to detect the presence of IL-13Rα1polypeptides and immunoaffinity chromatography to purify IL-13Rα1polypeptides. Further, those antibodies of the present invention thatcan inhibit signaling by IL-13 through the IL-13 receptor, as well asthose antibodies that can inhibit signaling by IL-13 and IL-4 throughthe IL-13 receptor, may be used to inhibit a biological activity thatresults from such signaling.

Therefore, in one embodiment, such antibodies may be used in therapeuticapplications to treat disorders caused or exacerbated (directly orindirectly) by the signaling of IL-13 or IL-4 through the IL-13 receptorcomplex. A therapeutic application involves in vivo administration of ablocking antibody to a mammal in an amount effective to inhibitsignaling by IL-13 and/or IL-4 through the IL-13 receptor. Preferably,the antibodies are human or humanized monoclonal antibodies of thepresent invention.

The antibodies may be used to treat diseases or conditions induced byeither or both IL-13 and IL-4 including but not limited to fibrosis,Hodgkin's disease, ulcerative colitis, scleroderma, lung disorders suchas asthma and chronic obstructive pulmonary disease, allergic rhinitis,oncological conditions, inflammatory bowel disease and otherinflammatory conditions in the gastrointestinal tract and allergicreactions to medication.

An antibody in accordance with the present invention is the murinemonoclonal antibody 1D9, and humanized forms of mAb 1D9.

The amino acid sequence of the variable region of the light chain of mAb1D9 is presented in SEQ ID NO: 27. The amino acid sequence for thevariable region of the heavy chain of mAb 1D9 is presented as SEQ IDNO:28 Amino acid sequence of murine 1D9 CDR regions from V_(L) domaingrafted onto a human consensus framework is presented in SEQ ID NO: 25Amino acid sequence of murine 1D9 CDR regions from V_(H) domain graftedonto human consensus framework is presented as SEQ ID NO: 26.

Antibodies of the present invention include, but are not limited to,monoclonal antibodies that comprise, in their light chain, residues 1 to112 of SEQ ID NO:25; and antibodies that additionally or alternativelycomprise, in their heavy chain, residues 1 to 121 of SEQ ID NO:26, ormonoclonal antibodies that comprise, in their light chain, residues 1 to112 of SEQ ID NO:27; and antibodies that additionally or alternativelycomprise, in their heavy chain, residues 1 to 121 of SEQ ID NO:28.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of mAb 1D9; a mAb that is cross-reactive with mAb 1D9;a mAb that binds to the same epitope as mAb 1D9; a mAb that competeswith mAb 1D9 for binding to a cell that expresses human IL-13Rα1; a mAbthat possesses a biological activity of mAb 1D9; and an antigen-bindingfragment of any of the foregoing antibodies. Antibodies in accordancewith this embodiment include 6A9 and 3F10 as discussed in the Examples.

In one embodiment, the antibody has a binding affinity for humanIL-13Rα1 that is substantially equivalent to the binding affinity of mAb1D9 for human IL-13Rα1. mAb 1D9 is an IgG1 antibody. mAb of otherisotypes (including but not limited to IgG4), derived from mAb 1D9 arealso encompassed by the present invention. Hybridoma cell lines thatproduce any such monoclonal antibodies also are provided by the presentinvention.

Procedures for switching (altering) the subclass or isotype of anantibody are also well known to those skilled in the art. Suchprocedures may involve, for example, recombinant DNA technology, wherebyDNA encoding antibody polypeptide chains that confer the desiredsubclass is substituted for DNA encoding the corresponding polypeptidechain of the parent antibody. This procedure is useful, for example, incertain antibody therapeutic applications where are particular antibodyisotope is preferred, such as in the treatment of asthma where IgG4 maybe the preferred antibody isotype.

One example of a biological activity of mAb 1D9 is the ability to bindto IL-13Rα1 and inhibit signaling by IL-13 and IL-4 through the IL-13receptor complex. In one embodiment, a mAb of the invention possessesIL-13 biological activity blocking activity substantially equivalent tothat of mAb 1D9; and possesses IL-4 biological activity blockingactivity substantially equivalent to that of mAb 1D9. Such activity maybe measured in any suitable conventional assay (e.g. as measured in theCD23 expression assay described below).

Particular embodiments of the invention are directed to novelpolypeptides. DNA and amino acid sequence information has beendetermined for polypeptides that are components of certain antibodies ofthe present invention, as discussed in Examples 7, 8, and 9 below. Amongthe polypeptides of the present invention is a purified polypeptidecomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence presented in SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27 and SEQ ID NO:28. For in vivo use, the polypeptides advantageouslyare purified. A polypeptide may be purified individually, or in the formof a purified antibody of which the polypeptide is a component.

The ability of the antibodies of the present invention to interfere withsignaling by IL-13 and/or IL-4 through the IL-13 receptor complex can beconfirmed in a number of assays.

One assay that may be used is described in International PatentPublication No. WO 01/92340, which is incorporated herein by reference.This assay is based on ability of both IL-13 and IL-4 to enhance theexpression of the activation-associated surface antigen CD23 on human Bcells. The antibodies of the present invention are tested for theability to inhibit CD23 expression induced by IL-13 and by IL-4.

In brief, antibodies raised against human IL-13Rα1 can be tested eitherin the form of hybridoma supernatants or purified protein. Prior toaddition to cultures, the antibodies are buffer exchanged againstculture medium (RPMI 1640 plus 10% v/v heat-inactivated fetal bovineserum) by centrifugation, using Centricon filter devices (Amicon) with a10 kDa cutoff.

Human peripheral blood B cells are purified as described (Morris et al.,J. Biol. Chem. 274: 418-423, 1999). The B cells (3×10⁵/well) in culturemedium are placed in 96-well round-bottomed microtiter plates andpreincubated at room temperature for 30 min with test antibodies.Recombinant human IL-13 or IL-4 is then added to the cultures, and thecells cultured for 20-24 hours at 37° C. in a humidified atmosphere of5% CO₂. At the end of the culture period, the cells are washed once inPBS+0.02% NaN₃ in the 96-well culture plate and resuspended in blockingbuffer (2% normal rabbit serum+1% normal goat serum in PBS+NaN₃).

Phycoerythrin (PE)-conjugated CD23 monoclonal antibody (mAb) orPE-conjugated isotype control mAb (both from Pharmingen) are added tocells at a final dilution of 1:10. Cells are incubated for 30 minutes at4° C., washed ×3 in PBS+NaN₃ and analyzed on a FacScan (BectonDickinson) for CD23 expression.

Negative controls such as cells cultured with hybridoma growth medium orisotype-matched non-blocking human anti-hIL-13 receptor antibody areincluded. An anti-huIL-4R murine mAb (R&D Systems), previously shown toblock the binding and function of both hIL-4 and hIL-13, can be used asa positive control for neutralization of CD23 induction by IL-4 andIL-13.

An alternative assay for identifying antibodies that function asIL-13Rα1 antagonists and block signaling by either IL-13 and/or IL-4 isdescribed below and in the Examples.

In this assay, 293A12-cells are engineered to express chimericpolypeptides comprising the extracellular domain of either IL-13Rα1 orIL-4Rα operably connected to the transmembrane and cytoplasmic domainsof the protein, gp130. When the engineered 293A12-cells are in thepresence of IL-13 or IL-4, the chimeric polypeptides form aheterodimeric receptor complex which permits signal transduction tooccur. The IL-13- or IL-4-mediated signal transduction is observable viaan identifiable signal, such as the activation of a gene encoding areporter molecule (Example 5).

Anti-IL-13Rα1 antibodies that antagonize IL-13 or IL-4 signaling throughthe IL-13 receptor will inhibit IL-13- and IL-4-mediated activation ofthe reporter molecule.

The level of signal transduction is conveniently determined by selectingcells wherein signal transduction activates a pathway regulating theexpression of a gene encoding a reporter molecule that provides anidentifiable signal. Preferred reporter molecules are enzymes such asluciferase.

293A12 cells are particularly preferred in this assay as they are 293Tcells which stably express genetic material encoding a luciferasereporter molecule (Example 3). The expression of the luciferase reportermolecule is regulated by a STAT-3 signaling pathway which is activatedby gp130 signaling.

The signal transduction portion from gp130 is particularly preferred, asit induces STAT-3 phosphorylation which leads to the expression of theSTAT-3 activated luciferase reporter gene. However, the signaltransduction portion from other molecules may also be employed. Thechoice of the signal transduction portion of the polypeptides must bematched to the activation or promoter portion of the gene encoding thereporter molecule.

Those skilled in the art appreciate that the cell based assays of theinvention, for example described above and in Example 4, may be utilisedas a basis for screening for modulators of IL-13Rα1/ligand interaction.While such methods are well known to those skilled in the art, a briefdescription of the method is provided herein. The method involvessubjecting appropriately engineered cells to a signal producing amountof IL-13 or IL-4 under conditions where, in the absence of anyantagonism of ligand receptor binding, a signal, for example luciferaseexpression, may be detected. The exposure is then conducted in thepresence of test compounds and the level of signal detected comparedwith that detected in the absence of a test compound. Test compounds mayinclude compound libraries, for example libraries of natural productextracts or libraries of synthetic compounds. Alternatively, phagedisplay libraries of antibody variable domains and the like, or panelsof monoclonal antibodies against IL-13Rα1 may be screened across theassay.

Chimeric polypeptides that may be used in the assay of the presentinvention are described in Examples 1 and 2 and comprise the amino acidsequences set forth in SEQ ID NO:8 and SEQ ID NO:10.

cDNA encoding the chimeric polypeptides contemplated for use in thisassay comprise a nucleotide sequence selected from SEQ ID NO:7 and SEQID NO:9. The sequence defined by SEQ ID NO:7 comprises a sequence whichencodes the IL-4Rα extracellular domain fused to the transmembrane andcytoplasmic domains of gp130. SEQ ID NO:9 comprises a sequence whichencodes the IL-13Rα1 extracellular domain fused to the transmembrane andcytoplasmic domains of gp130.

Although 293A12 cells are described in the assay of the presentinvention, other cells may be used. Generally a eukaryotic cell isemployed, and more particularly, a mammalian cell. The mammalian cellsmay be derived from humans, livestock animals, laboratory test animalsand companion animals. Non-mammalian cells contemplated herein includecells from avian species, reptilian species, amphibian species andinsect species. Preferably, the cell lacks endogenous γc.

The term “operably connected” is used in its broadest context to includemolecules which have associated together such that they are infunctional interaction with each other. Generally, the association is bya chemical linkage or bond. Preferably, the chemical linkage or bond isa peptide bond. The terms include, therefore, a polypeptide comprising acontiguous series of amino acids each linked via a peptide bond whereinone contiguous series of amino acids has ligand-binding properties andanother contiguous series of amino acids has signal transductionproperties.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, agents used for adjusting tonicity, buffers, chelating agents,and absorption delaying agents and the like. The use of such media andagents for pharmaceutical active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active ingredient, use thereof in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dilution mediumcomprising, for example, water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of superfactants. The preventions ofthe action of microorganisms can be brought about by variousanti-bacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include agents to adjust tonicity, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin. The compositions may also include buffers andchelating agents.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze-drying technique which yield a powder ofactive ingredient plus any additionally desired ingredient.

The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained.

The compositions of the present invention are useful in modifying anIL-13- or IL-4-mediated condition including but not limited to fibrosis,Hodgkin's disease, ulcerative colitis, scleroderma, lung disorders suchas asthma and chronic obstructive pulmonary disease, allergic rhinitis,oncological conditions, inflammatory bowel disease and otherinflammatory conditions in the gastrointestinal tract, allergicreactions to medication and any other IL-13 mediated diseases orconditions.

The human and humanized antibodies of the present invention and inparticular humanized 1D9 are useful in the treatment of such conditions.Any adverse condition resulting from IL-13 and/or IL-4 interaction withIL-13Rα1 may be treated or prevented by the administration of theantibodies of the invention such as humanized 1D9.

Accordingly, another aspect of the present invention contemplates amethod for the treatment or prophylaxis of a condition mediated by IL-13and/or IL-4 such as but not limited to an inflammatory condition, saidmethod comprising administering to a subject an effective amount of anantibody, such as humanized 1D9, for a time and under conditionssufficient to inhibit IL-13 and/or IL-4 signaling through the IL-13receptor complex.

An “effective amount” in this context is an amount of an antibodysufficient to reduce IL-13 and/or IL-4 signaling through the IL-13receptor complex by at least 40%, preferably at least 50%, morepreferably by at least 60%, still more preferably by at least 70-80% orgreater than 90%.

The method may also be measured at the level of amelioration ofsymptoms. Hence, an effective amount would be that amount required to atleast partially alleviate symptoms of, for example, inflammation.

Preferably, the subject is a human. However, veterinary applications arealso contemplated for livestock animals as well as companion animals. Insuch cases it would be necessary to prepare an appropriate antibodydesigned to avoid an immunogenic response to the antibody by the mammal.

In a specific embodiment, therefore, the present invention provides amethod for ameliorating the effects of IL-13 or Il-4 mediated conditionsin a human subject, said method comprising administering to said subjectan effective amount of a humanized 1D9 monoclonal antibody or itsequivalent for a time and under conditions sufficient to ameliorate theeffects of inflammation.

The present invention further contemplates the use of a humanized 1D9 orits equivalent in the manufacture of a medicament in the treatment orprophylaxis of an inflammatory condition in a subject.

The humanized 1D9 may also be used to deliver specific drugs conjugatedthereto to particular sites, such as cells carrying the IL-13Rα1receptor. The humanized 1D9 antibodies may also be used to conductimaging analysis to screen for active IL-13Rα1 receptors.

The present invention is further described by the following non-limitingExamples.

Example 1 Construction of the IL13Rαd/gp130 Chimera

To generate the chimeric IL13Rα1/gp130 cDNA molecule, the IL13R wasamplified with a 5′ oligomer containing an Asc1 restriction enzyme site,for cloning into the pEFBOS vector, and a 3′ oligomer that contained anoverlapping region homologous to the gp130 cDNA. The oligomers used toamplify the gp130 cDNA comprised a 3′ oligomer containing an Mlu1restriction enzyme site.

IL-13R1 Oligomers

5′ oligomer: AGCTGGCGCGCCAGGCGCCTACGGAAACTCAGCCACCTGTG  [SEQ ID 11] 3′oligomer: CAGGCACGACTATGGCTTCAATTTCTCCTGTGGAATTGCGCTTCTTACCTATACTC[SEQ ID NO: 12]gp130 Oligomers

5′ oligomer: [SEQ ID NO: 13] GGAGAAATTGAAGCCATAGTCGTGCCTGTTTGCTTAGC 3′oligomer: [SEQ ID NO: 14] ACGTACGCGTTCACTGAGGCATGTAGCCGCCTTGCCG

The PCR conditions to amplify the IL-13Rα1 and the gp130 regionsrequired for the construction of the chimeric cDNA were identical forboth molecules. One cycle of 94° C. for 2 mins, 35 cycles of 94° C. for10 secs, 50° C. for 10 secs and 68° C. for 1 min and one cycle at 68° C.for 5 mins. The molecules were amplified using the PLATINUM Pfx DNApolymerase kit (Invitrogen).

The chimeric cDNA molecule was amplified using the PCR productsgenerated from the previously described reactions, with the sameconditions being used, except that the extension time was lengthenedfrom 60 to 90 secs. The oligomers used to generate the chimeric cDNAmolecule were:

5′ oligomer: [SEQ ID NO: 11] AGCTGGCGCGCCAGGCGCCTACGGAAACTCAGCCACCTGTG3′ oligomer: [SEQ ID NO: 14] ACGTACGCGTTCACTGAGGCATGTAGCCGCCTTGCCG

The chimeric cDNA was the cloned into the Mlu1 restriction enzyme siteof the pEFBOS mammalian expression vector, which contains the murineIL-3 signal sequence and a FLAG peptide at the N terminus. The cloningwas carried out using the Amersham ligation kit.

Example 2 Construction of the IL-4Rα Igp130 Chimera

The IL-4Rα was amplified by RT-PCR, from mRNA isolated from Jurkatcells, using the Titan RT-PCR kit (Roche). The oligomers use to amplifythe IL-4Rα were:—

5′ oligomer: [SEQ ID NO: 15] TGA AGG TCT TGC AAG AGC CCA CCT GCG 3′oligomer: [SEQ ID NO: 16] GTG CTG CTC GAA GGG CTCCCT GTA GGA G

The PCR conditions were as follows. One cycle of 50° C. for 30 mins and94° C. for 2 mins, 35 cycles of 94° C. for 30 secs, 50° C. for 30 secsand 68° C. for 1 min and one cycle of 68° C. for 7 min.

To generate the chimeric IL-4Rα/gp130 cDNA molecule, the IL-4Rα wasamplified with oligomers that comprised of a 5′ oligomer that containedan Asc1 restriction enzyme site, for cloning into the pEFBOS vector anda 3′ oligomer that contained an overlapping region homologous to thegp130 cDNA. The oligomers used to amplify the gp130 cDNA comprised a 3′oligomer containing an Mlu1 restriction enzyme site.

IL-4R Oligomers

5′ oligomer: AGCTGGCGCGCCTGAAGGTCTTGCAGGAGCCCACCTGCG  [SEQ ID NO: 17] 3′oligomer: CAGGCACGACTATGGCTTCAATTTCTCCGTGCTGCTCGAAGGGCTCCCTGTAGGAG[SEQ ID NO: 18]gp130 Oligomers

5′ oligomer: [SEQ ID NO: 13] GGAGAAATTGAAGCCATAGTCGTGCCTGTTTGCTTAGC 3′oligomer: [SEQ ID NO: 14] ACGTACGCGTTCACTGAGGCATGTAGCCGCCTTGCCG

The PCR conditions to amplify the IL-4-a receptor and the gp 130 regionsrequired for the construction of the chimeric cDNA were identical forboth molecules. One cycle of 94° C. for 2 mins, 35 cycles of 94° C. for10 secs, 50° C. for 10 secs and 68° C. for 1 min and one cycle at 68° C.for 5 mins The molecules were amplified using the PLATINUM Pfx DNApolymerase kit (Invitrogen).

The chimeric cDNA molecule was amplified using the PCR productsgenerated from the previously described reactions, with the sameconditions being used, except that the extension time was lengthenedfrom 60 to 90 secs. The oligomers used to generate the chimeric cDNAmolecule were:

5′ oligomer: [SEQ ID NO: 17] AGCTGGCGCGCCTGAAGGTCTTGCAGGAGCCCACCTGCG 3′oligomer: [SEQ ID NO: 14] ACGTACGCGTTCACTGAGGCATGTAGCCGCCTTGCCG

The chimeric cDNA was cloned into the Mlu1 restriction enzyme site ofthe pEFBOS mammalian expression vector, which contains the murine IL-3signal sequence and a FLAG peptide at the N terminus. The cloning wascarried out using the Amersham ligation kit.

Example 3 Generation of A12 Cells

293T cells (obtained from Amrad Biotech) were cotransfected with 10 μgAPRE-luc (Nakajima et al., EMBO J. 15: 3651-3658, 1996) and 1 μgpGK-puro using lipofectamine (Life Technologies, Lot #KE4Y01).

Cells were selected in 25 μg/ml puromycin and positive clones tested forluciferase response.

Cell line A25-20 was subsequently further cloned by limit dilution,giving the clone 293T-A12.

Example 4 Development of Assays for Analysis of IL-13Rα1 Interaction

Human factor-dependent (GM-CSF, IL-6, IL-4, or IL-13 etc.) TF-1 cellswere previously used as the standard bioassay for IL-13 activity whichis based on assessing the neutralizing/inhibitory activity of mouse andhuman mAbs. However, the assay has proven to be extremely unreliablewith a relatively poor response to IL-13 and a low signal to backgroundratio.

Development of a Cell-Based Assay

The inventors developed an assay based on a chimeric receptor strategy.The strategy involves fusing the extracellular domain of both theIL-13Rα1 and the IL-4Rα to the transmembrane and cytoplasmic domains ofgp130. Following production of these two chimeric receptors in the293A12 cell line (a 293T derivative with stable expression of aluciferase reporter under the control of a STAT-3 responsive promoter),IL-13 mediated dimerization activates STAT-3 and subsequently luciferasereporter gene expression (FIG. 1).

An important aspect of this strategy is that it allows theidentification of IL-13Rα1 antagonists such as mAbs that inhibit IL-4signaling mediated through the IL-4 type II receptor complex. IL-4signals through a type I receptor complex that incorporates the IL-4Rαand γc, and a type II receptor complex that incorporates the IL-4Rα andIL-13Rα1. Cell lines such as TF-1 are not suited to this purpose as theyco-express γc and IL-13Rα1 such that IL-4 may signal through either ofthe two receptor complexes. In contrast, in the engineered cell line ofthe present invention, only IL-4 signaling through the type II complexshould lead to luciferase expression, irrespective of 293T cell γcexpression.

Using IL-13Rα1 and gp130 cDNAs as template, a human IL-13Rα1-gp130chimeric receptor cDNA is generated by splice-overlap-extension PCR andcloned into pEFBOS for expression as an N-terminal FLAG-tagged protein.For generation of the IL-4Rα-gp130 chimeric receptor, an IL-4Rα cDNA(extracellular domain only) is cloned by RT-PCR using mRNA extractedfrom TF-1 cells. The chimeric IL-4Rα-gp130 receptor cDNA is generated bysplice-overlap-extension PCR and also cloned into pEFBOS for expressionas an N-terminal FLAG-tagged protein.

Details of both chimeric receptors are provided in schematic form inFIG. 2. Transient expression in COS cells, followed by Western blotanalysis with anti-FLAG or anti-IL-13Rα1 antibodies confirmed that bothconstructs encode a protein of the expected molecular weight (FIG. 3).

To isolate stable lines, 293A12 cells are co-transfected with thechimeric receptor constructs and a vector encoding the gene forhygromycin resistance. Following hygromycin selection, 100 isolatedresistant colonies are picked and expanded through 48 and 24 wellplates. Subsequently 56 of the picked colonies are assayed forluciferase in the presence of LIF (+ve control), IL-13 and IL-4.Thirteen of the 56 colonies assayed appear to express luciferase inresponse to both IL-13 and IL-4 in addition to LIF (Table 2) and ofthese 11 were expanded for freezing and further analysis.

The two cell lines with the best signal to noise ratio (3.1.2 and 3.2.4)were subsequently cloned by limited dilution and for both, a full doseresponse analysis with respect to IL-4, IL-13 and LIF was conducted(FIG. 4). For both cell lines, the response to IL-13 appears similar tothat observed for LIF with 50% of maximal activity observed at 100-200pg/ml. For IL-4, 50% of maximal activity observed at 2-4 ng/ml for bothlines. Consistent with earlier data, the signal to noise ratio for bothlines is in excess of 10. The data indicate that these cell linesrepresent the best cell-based assays for either IL-13 or IL-4.

Molecular Assay

A molecular assay based on the interaction of IL-13Rα1 with IL-13represents the best primary screen for both monoclonal antibodies and,potentially, small molecule antagonists. As stated above, however, theinteraction of IL-13 with the IL-13Rα1 is weak (>200 nM) and notamenable to a simple ELISA-based approach. While FRET and fluorescencepolarization-based assays have been contemplated, the development ofsuch assays is labour and material intensive.

A chimeric receptor protein that incorporates the extracellular domainof the IL-13Rα1 (human or mouse) and the Fc portion of human IgG hasbeen developed (R & D Systems). These chimeric proteins are expressed aspreformed dimers, based on inter-Fc region disulphide bonds and areexpected to associate more tightly with IL-13 than the monomeric form ofthe receptor.

For initial Biosensor studies, human IL-13 was immobilized to theBiosensor chip and a dose-response analysis of human and mouseIL-13Rα1-Fc binding was completed. Both chimeric receptors associatedwith human IL-13, with the signal obtained for the mouse receptorsubstantially higher than that obtained with the human receptor. Similarresults are obtained with immobilized mouse IL-13. These findingsconfirm the cross-species activity of IL-13. To confirm the specificityof this interaction, a competitive binding-based approach is employed. Afixed concentration of chimeric mouse receptor protein was incubatedwith titrating soluble mouse IL-13 and binding of the receptor toimmobilized mouse IL-13 was assessed. The soluble IL-13 was able tocompete for binding to the chip in a dose-dependant manner. Similar datawas obtained using the chimeric human receptor.

A qualitative comparison of sensorgrams obtained in this study to dataobtained previously with monomeric receptor protein, indicated asubstantial improvement in binding kinetics. This improvement isattributed to a much slower off-rate for the dimeric form, compared withthe monomeric form, of the receptor. To further quantify thisinteraction a complete dose-response analysis using both human and mousechimeric receptor proteins and immobilized human and mouse IL-13 wasundertaken. Primary data obtained for the binding of the chimeric humanand mouse receptors to mouse IL-13 are presented in Table 3. Thechimeric mouse receptor appears to have an approximately 10-fold greateraffinity for both human and mouse IL-13 compared with the chimeric humanreceptor. Nevertheless, the chimeric human receptor demonstrates a100-fold increase in affinity for IL-13 compared with the monomeric formof the receptor.

Biosensor data indicate a substantial increase in binding affinity forthe dimeric form of the receptor compared with the monomeric form andsuggested that an ELISA-based approach to a molecular assay may befeasible. Preliminary experiments indicated that the interaction ofsoluble chimeric receptors with plate bound mouse IL-13 is readilydetectable using an anti-hulg-HRPO conjugate. As expected, a higherconcentration of the human receptor is required to obtain a signalequivalent to that obtained with the mouse receptor. Subsequently, bothchimeric mouse and human receptors were titrated over variousconcentrations of plate bound IL-13 to establish optimal assayconditions. Results indicated that the chimeric human receptor titratesover a dose-range of 0.312-10 μg/ml with plate bound IL-13 atconcentrations greater than 2.5 μg/ml. In comparison, the chimeric mousereceptor titrates over a dose-range of 0.02-0.625 μg/ml with plate boundIL-13 at greater than 1.25 μg/ml. As expected, control chimericreceptor, Flt-Fc, failed to bind in this assay.

Example 5 Analysis of IL-13Rα1-Specific Mouse mAbs Analysis UsingBiochemical Assays—Biosensor and ELISA

Initially mouse mAb 1D9 is tested for its ability to inhibit theinteraction of the chimeric human and mouse IL-13Rα1-Fc with IL-13 usingboth an ELISA- and Biosensor-based approach. In Biosensor studies, 1D9clearly inhibits the interaction of the chimeric human receptor withboth human and mouse IL-13 but has no effect on the binding of thechimeric mouse receptor (FIG. 5). Identical results are obtained withthe ELISA-based assay. 1D9 is a potent inhibitor of the chimeric humanreceptor, compared with a control mAb, but has no effect on the bindingof the chimeric mouse receptor to mouse IL-13 (FIG. 6). The Biosensorstudy incorporated a 1D9 dose-response analysis and a furtherdose-response analysis was undertaken using the ELISA. These resultsdemonstrated that 1D9 is a potent antagonist with an IC₅₀ similar to theconcentration of target receptor used in the assays (−20 nM for theELISA). The selectivity of 1D9 for human but not mouse IL-13Rα1 is alsodemonstrated using Western blot analysis.

In further studies, additional mouse mAbs are tested by ELISA for theirability to inhibit the interaction of the chimeric human receptor withIL-13. mAb 6A9, which interacts with the same epitope as 1D9 showspotent antagonist activity (FIG. 7). mAb 3F10 binds to a differentepitope and appeared to have a partial inhibitory activity. In contrast,mAb 2A2 which binds to a further unrelated epitope and which is mostuseful in Western blot analysis, fails to inhibit the chimericreceptor-ligand interaction. As expected unrelated control mAbs 2H10 and6C12 had no effect on binding.

Analysis Using the Cell-Based Assay

The uncloned IL-13/IL-4-responsive transfected 293A12 derivative, 3.2.4,is expanded and used to assess the antagonist activity of theIL-13Rα1-specific mouse mAbs 1D9, 6A9 and 2A2. 3.2.4 cells arepre-incubated for 45 mins in titrating mAb prior to the addition ofeither IL-13 or IL-4 to a final concentration of 10 or 1 ng/ml.Luciferase production is assessed at 24 hrs.

Results presented in FIG. 8 demonstrate that, in agreement withbiochemical assay data, mAbs 1D9 and 6A9 (but not mAb 2A2) are able toinhibit IL-13 mediated luciferase expression. For both 6A9 and 1D9, theinhibitory activity was most pronounced with IL-13 at 1 ng/ml. 1D9appeared to be more potent than 6A9 with almost complete inhibition ofthe response to 1 ng/ml of IL-13 over the dose-range of mAb tested. Thenegative control unrelated mAb 2H10 had no effect on IL-13-inducedluciferase expression as expected.

Unlike biochemical-based assays and existing cell-based assays, the3.2.4 line allows the effects of IL-13Rα1 specific mAbs on IL-4signaling through the type II IL-4 receptor complex to be assessed.Results presented in FIG. 9 demonstrate that both mAbs that are able toinhibit IL-13-mediated activity are also able to inhibit IL-4 mediatedluciferase expression. Again, the effect was substantially morepronounced with cytokine at 1 ng/ml compared with 10 ng/ml and again 1D9appeared to be the most potent of the two antibodies. As with IL-13,neither mAb 2A2 nor the negative control mAb 2H10, had any effect onIL-4-induced luciferase expression.

Example 6 Cloning and Sequencing of the Murine Antibody Variable Regions

Messenger RNA was prepared from hybridoma cells producing the 1D9 mAband reverse transcribed using an oligo-dT primer to produce cDNA.Partially degenerate PCR primers based on the amino-terminal amino acidsequence and the antibody isotype were used to amplify the mature mouseheavy and light variable domains and incorporate restriction enzymesites for cloning. The subsequent clones and PCR products were sequencedto reveal the amino acid sequence for each of the variable regions of1D9 (FIG. 1).

Example 7 Construction of a Human Fab Template

A synthetic human fragment antibody binding (Fab) was generated fromsynthetic oligonucleotides as a template for intermediate and humanizedvariants of the 1D9 mouse antibody. The synthetic human Fab consisted ofvariable domain sequences derived from the consensus sequences for themost abundant human subclasses (V_(L)κ subgroup I and V_(H) subgroupIII) and human constant regions (REI human κ₁ light chain C_(L) and IgG1C_(H)1). The synthetic human Fab sequences were subsequently insertedinto a single E. coli expression vector to generate a dicistronicconstruct for expression of either soluble or phage displayed functionalFab.

Example 8 Generation of CDR-Grafted Fabs and Mouse-Human Chimeric Fabs

As a starting point for humanization, a CDR-grafted Fab was generated bygrafting the six complementarity-determining regions (CDRs) of theparent 1D9 antibody onto the synthetic human Fab. Optimization of keyframework residues within a CDR-graft Fab is often required for correctpresentation of the murine CDRs by the human framework and henceretention of potent binding affinity. Chimeric Fab fragments areequivalent in their antigen binding properties to the fully murine Fabfragment so can be used to determine if the CDR-grafted Fab requiresframework optimization. A mouse-human chimeric Fab fragment consistingof the murine 1D9 heavy and light chain variable regions fused to thecorresponding synthetic human constant domains was therefore generatedas a reference for antigen binding affinity.

Example 9 Comparison of the Binding Affinities of the Chimeric andCDR-Grafted Fabs

The binding affinity of the CDR-grafted and chimeric Fabs forIL-13R.alpah.1 were compared in Competition based assays, both as phagedisplayed Fabs in an ELISA format (FIG. 11A) and as purified solubleprotein by a BIACORE™ biosensor competition assay (FIG. 11B). TheCDR-grafted Fab has similar affinity for IL-13R.alpha.1 as the referencemurine-human chimeric Fab. This indicates that the CDR-graft Fab doesnot require optimization of the framework residues and can be consideredhumanized.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 2 Response of transfected (FLAG-tagged IL-13Rα1-gp130 andIL-4Rα-gp130 and picked 293A12 colonies to LIF, IL-13 and IL-4 Line# MedLIF* IL-13 IL-4 3.1.1 6791 61220 7381 12469  3.1.2 3539 42150 34094(9.6)  53998 (15.2) 2.3.1 4626 43264 4383 4458 2.3.2 5850 52813 53775252 1.2.2 4921 45047 15093 (3.1) 29866 (6.1) 1.2.3 7222 159076 71837298 3.2.4* 7783 61163 42046 (5.4) 117971 (15.1) 3.2.5 6823 62906  73145(10.7) 129369 (18.9) 3.2.6 7849 67302 8307 16826  3.2.7 21589 16310288581 (4.1) 136760 (6.3)  3.2.8 10698 89447 10352  12778  3.2.9 409345747 4141 4530 *LIF, IL-13 and IL-4 all used at a final concentrationof 100 ng/ml, 24 hr assay. *Representative data, 12 of 56 coloniesassessed.

TABLE 3 Affinity (KD) of chimeric mouse and human IL-13Rα1-Fc proteinsfor immobilized mouse and human IL-13 Chimeric receptor* mIL-13Rα1-FchIL-13Rα1-Fc Mouse IL-13 0.536 nM 15.11 nM Human IL-13 0.784 nM  5.93 nM

BIBLIOGRAPHY

-   Ahdieh et al., Am J. Physiol. Cell Physiol. 281(6): C2029-2038, 2000-   Akaiwa et al., Cytokine 13: 75-84, 2001-   Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988-   Bailer et al., Eur. J. Immunol. 30(5): 1340-1349, 2000-   Bailer et al., J. Immunol. 162(12): 7534-7542, 1999-   Bird, Science 242: 423, 1988-   Callard et al., Immunology Today 17(3): 108, 1996-   Danahay et al., Am. J. Physiol. Lung Cell Mol. Physiol. 282(2):    L226-236, 2002-   David et al., Oncogene 20(46): 6660-6668, 2001-   Gauchat et al., Eur. J. Immunol. 28: 4286-4298, 1998-   Gauchat et al., Eur. J. Immunol. 30: 3157-3164, 2000-   Howard et al., Am J Hum Genet 70(1): 230-236, 2002-   Howard et al., Am. J. Hum. Genet. 70(1): 230-236, 2002-   Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879, 1988-   Kabat et al. in Sequences of Proteins of Immunological Interest, 5th    Ed., US Dept. of Health and Human Services, PHS, NIH, NIH    Publication No. 91-3242, 1991-   Kortt et al., Protein Engineering 10: 423, 1997-   Larrick et al., Bio/Technology 7: 934, 1989-   Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439, 1987-   Monoclonal Antibodies, Hybridomas: A New Dimension in Biological    Analyses, Kennet et al. (eds.), Plenum Press, New York, 1980-   Morris et al., J. Biol. Chem. 274: 418-423, 1999-   Morse et al., Am. J. Physiol. Lung Cell Mol. Physiol. 282(1):    L44-49, 2002-   Noguchi et al., Hum Immunol 62(11): 1251-1257, 2001-   Perez et al., J. Immunol. 168(3): 1428-1434, 2002-   Riechmann et al., Nature 332: 323, 1988-   Ward et al., Nature 334: 544, 1989-   Winter and Harris, TIPS 14: 139, 1993

1. An antibody or antigen-binding fragment thereof which binds to amammalian IL-13Rα1 chain or an antibody-binding portion thereof, whereinthe binding of the antibody to IL-13Rα1 antagonizes IL-13receptor-mediated signaling.
 2. The antibody of claim 1 wherein theIL-13 receptor-mediated signaling is by IL-13 and IL-4.
 3. The antibodyof claim 1 wherein the antibody is a monoclonal antibody.
 4. Theantibody of claim 3 wherein the IL-13Rα1 is of human origin.
 5. Theantibody of claim 4 wherein the antibody is a human antibody.
 6. Theantibody of claim 4 wherein the antibody is a deimmunized antibody. 7.The antibody of claim 6 wherein the antibody is a humanized antibody. 8.The antibody of claim 1 wherein the antibody is a fragment of a wholeantibody.
 9. The antibody of claim 8 wherein the antibody fragment is anFv, Fab, Fab′ or F(ab′)₂ fragment.
 10. An antibody comprising a variableregion of a light chain of at least one CDR from the light chain of anantibody of claim
 1. 11. The antibody of claim 10 wherein the variableregion is from murine monoclonal antibody 1D9 deposited at the EuropeanCollection of Cell Cultures (ECACC), Centre for Applied Microbiology andResearch, Porton Down, Salisbury, United Kingdom, under Accession No.03032101.
 12. The antibody of claim 10 or 11 comprising a CDR as definedin any one or more of SEQ ID NOs: 19 to
 21. 13. The antibody of claim 10wherein the variable region is defined by SEQ ID NO:
 27. 14. An antibodycomprising a variable region of a heavy chain of at least one CDR fromthe light chain of an antibody of claim
 1. 15. The antibody of claim 14wherein the variable region is from murine monoclonal antibody 1D9deposited at the European Collection of Cell Cultures (ECACC), Centrefor Applied Microbiology and Research, Porton Down, Salisbury, UnitedKingdom, under Accession No.
 03032101. 16. The antibody of claim 14 or15 comprising a CDR as defined in any one or more of SEQ ID NOs: 22 to24.
 17. The antibody of claim 14 wherein the variable region is definedby SEQ ID NO:
 28. 18. A method of treating a disease condition in amammal comprising administering to said mammal an effective amount of anantibody of any one of claim 1, 10, or
 14. 19. The method of claim 18wherein the mammal is a human.
 20. The method of claim 19 wherein thedisease condition is fibrosis, Hodgkin's disease, ulcerative colitis,scleroderma, allergic rhinitis, oncological conditions, a lung disorderor an inflammatory disorder.
 21. The method of claim 20 wherein the lungdisorder is asthma or chronic obstructive pulmonary disease.
 22. Themethod of claim 20 wherein the inflammatory condition is a condition ofthe gastrointestinal tract
 23. A method for the treatment or prophylaxisof a condition mediated by IL-13 and/or IL-4 such as but not limited toan inflammatory condition, said method comprising administering to asubject an effective amount of an antibody, such as humanized 1D9, for atime and under conditions sufficient to inhibit IL-13, or IL-13 and IL-4signaling through the IL-13 receptor complex.
 24. The method of claim 23wherein the mammal is a human.
 25. An isolated monoclonal antibody or anantigen-binding fragment thereof which competes with monoclonal antibody1D9 for binding to the IL-13Rα1 chain.